1. Technical Field
The present invention relates generally to DNA hybridization probes, and more particularly relates to a stable, covalent conjugate of an oligonucleotide and horseradish peroxidase (HRP).
2. Description of the Prior Art
Non-isotopically labelled synthetic DNA fragments have found broad application in molecular biology--e.g., in the areas of DNA sequencing, DNA probe-based diagnostics, and the like. The conjugate disclosed herein is prepared using reagents which facilitate the labeling of oligonucleotides with specific groups by incorporating one or more modifiable sulfhydryl groups at one or more hydroxyl sites within the oligonucleotide. These "functionalizing" reagents are described in co-pending application Serial No. 104,200, of common inventorship and entitled "Oligonucleotide Functionalizing Reagents and Methods", filed on even date herewith. The disclosure of that application is hereby incorporated by reference in its entirety.
Methods of introducing a sulfhydryl group at the 5' terminus of synthetic oligonucleotides are known. For example, Connolly, in Nuc. Acids Res. 13(12) 4485-4502 (1985) and in Nuc. Acids Res. 15(7):3131-3139 (1987), describes a method of incorporating a sulfhydryl moiety into synthetic DNA using S-trityl-0-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol--i.e., reagents given by the formula ##STR1## where x is 2, 3 or 6. Connolly further describes derivatization of the sulfhydryl-containing oligonucleotides with thiol-specific probes.
However, this and other prior art methods suffer from a number of disadvantages as discussed in co-pending application Serial No. 104,200, incorporated by reference above.
As discussed therein, there is a need in the art for oligonucleotide functionalizing reagents which address those considerations. The present invention is directed to a method of "derivatizing" the sulfhydryl-functionalized oligonucleotides described in application Ser No. 104,200, supra, give covalent HRP conjugates.
Covalent conjugates of oligonucleotides and labelling enzymes have been described in the literature. For example, Jablonski et al., in Nuc. Acids Res. 14(15):6115-6128 (1986), describe covalent conjugates of alkaline phosphatase and oligonucleotides prepared using the homobifunctional reagent disuccinimidyl suberate. Renz and Kurz, in Nuc. Acids Res. 12(8):3435-3445 (1984), describe a covalent complex of HRP and oligonucleotides using a polyethyleneimine spacer chain having a molecular weight of about 1400. Also, Ruth and Jablonski, in Nucleosides and Nucleotides 6(1&2):541-542 (1982), disclose conjugates of oligodeoxynucleotides and alkaline phosphatase having a 19-atom spacer chain between the oligomer and the enzyme. While these probes have been used successfully, it would nevertheless be desirable to provide probes which are more stable and which generate color faster, thus yielding a more effective and more readily monitorable means of detection.